Parasitology International 63 (2014) 591–596

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Baylisascaris potosis n. sp., a new ascarid nematode isolated from captivekinkajou, Potos flavus, from the Cooperative Republic of Guyana

Toshihiro Tokiwa a, Shohei Nakamura b, Kensuke Taira b, Yumi Une a,⁎a Laboratory of Pathology, School of Veterinary Medicine, Azabu University, Kanagawa, Japanb Laboratory of Parasitology, School of Veterinary Medicine, Azabu University, Kanagawa, Japan

⁎ Corresponding author. Tel./fax: +81 42 769 1628.E-mail address: une@azabu-u.ac.jp (Y. Une).

http://dx.doi.org/10.1016/j.parint.2014.03.0031383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 December 2013Received in revised form 6 March 2014Accepted 14 March 2014Available online 22 March 2014

Keywords:Baylisascaris potosis n. sp.KinkajouPotos flavusScanning electron microscopyCOX128S rDNA

Wedescribe a newnematode species, Baylisascaris potosis n. sp., isolated from captive kinkajou, Potos flavus, fromthe Cooperative Republic of Guyana. The nematodewas found in fecal specimens, identifiedmorphologically, andconfirmed genetically. The new species is similar to Baylisascaris procyonis, Baylisascaris columnaris, and otherBaylisascaris species, but is distinguished by the position of themale phasmidial pole. Nuclear andmitochondrialDNA sequence analyses confirmed that the new species is phylogenetically distinct from all the members of thegenus Baylisascaris, and groups with B. procyonis and B. columnaris. This nematode is the 10th species assigned tothe genus Baylisascaris.

© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Baylisascaris Sprent 1968 (Nematoda: Ascarididae) is a smallgenus with only nine recognized species; namely, Baylisascaristransfuga (Rudolphi, 1819), Baylisascaris laevis (Leidy, 1856),Baylisascaris columnaris (Leidy, 1856), Baylisascaris melis (Gedoelst,1920), Baylisascaris schroederi (McIntosh, 1939), Baylisascaris procyonis(Stefanski and Zarnowski, 1951), Baylisascaris devosi (Sprent, 1952),Baylisascaris ailuri (Wu et al., 1987), and Baylisascaris tasmaniensisSprent, 1970 [1,2]. Members of the genus Baylisascaris occur primarilyin placental carnivores, with B. tasmaniensis and B. laevis occurring inmarsupial carnivores and rodents, respectively. Although the raccoonroundworm B. procyonis is recognized as a cause of serious or fatallarva migrans in humans and animals [3,4], other species of the genusBaylisascaris are considered potential zoonotic nematodes [5].

The kinkajou, Potos flavus (Chordata: Procyonidae), is a medium-sized (1.4–4.5 kg), nocturnal, and arboreal mammal native in the low-land rainforests of South and Central America [6]. Previous studieshave shown kinkajous to be a definitive host for B. procyonis, the rac-coon roundworm [7–9], and morphology of the nematodes identifiedto B. procyonis isolated from kinkajous in Colombia was reported byOverstreet [7]. Classification of species of the Baylisascaris and the phy-logenetic relationships among members of the genus have been pro-posed based on morphological characters [1,10]. Recently, molecular

.

studies have demonstrated that the second internal transcribed spacer(ITS2), 28S nuclear ribosomal DNA (rDNA), and mitochondrial cyto-chrome oxidase subunit I (COX1) can provide genetic markers forspecies-level identification of the genus Baylisascaris [11–13]. However,phylogenetic analyses using the ribosomal ITS2 region indicatedthat Baylisascaris sp. from a captive kinkajou were different fromB. procyonis from raccoons, in spite of morphological similarities [8].These data suggest that nematodes from kinkajous should be consid-ered to be cryptic species of the genus Baylisascaris, being only distin-guished by molecular based analyses.

In 2013, we collected fecal samples from two captive kinkajous andfound Baylisascaris eggs and nematodes. Considering the importance ofB. procyonis and othermembers of the genus in public health, taxonomicposition of Baylisascaris from kinkajou should be revealed. In this study,we characterized nematodes of the genus Baylisascaris collected fromtwo captive kinkajous by morphology and molecular based analysesand described Baylisascaris potosis n. sp.

2. Materials and methods

2.1. Collection of materials

The wild female kinkajous, more than two years old, born in the Co-operative Republic of Guyana were imported to Japan in December2012. Nematodes and eggs were obtained from fresh fecal samplesfrom these kinkajous. Specimens were washed in phosphate bufferedsaline (pH 7.2), fixed in 10% (v/v) neutral-buffered formalin solution

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and stored in the dark for later study. A body segment was preserved in100% ethanol and stored at −30 °C until used for molecular analyses.Type specimens were deposited in the National Museum of Natureand Science, Tokyo, Japan.

2.2. Morphology and morphometric analyses

Nematodes were cleared with lactophenol solution (25% glycerin,25% lactic acid, 25% phenol, and 25% distilled water) in a petri dish,observed using a binocular stereo microscope (Nikon, Japan), and illus-trated by the aid of drawing tube fitted to an eyepiece. Eggs were ob-served by using a Nikon Eclipse E600 microscope (Nikon, Japan). Theesophago-intestinal region of worm was fixed in 10% neutral-bufferedformalin, embedded in paraffin, sectioned transversally, and stainedwith hematoxylin and eosin (HE). Measurements were taken with anocular micrometer and represented in mm as a mean followed by theranges in parentheses. For scanning electron microscopy, formalin-fixed samples were washed three times with distilled water and post-fixed in 2% (w/v) osmium tetraxide aqueous solution for 2 h at roomtemperature. After three washes with phosphate buffer (pH 7.2), thesamples were dehydrated twice through an ethanol series (50–99%)and three times in 100%, keeping for 30 min in each concentration.The samples were then immersed in t-butyl alcohol (Wako, Japan) for30 min at −30 °C. The samples were freeze-dried using a JFD-310freeze dryer (JEOL, Japan), sputter-coated with platinum–palladium ina JFC1600 sputter–coater (JEOL), and observed using a JSM-6380-LVscanning electron microscope (JEOL) at the Department of VeterinaryParasitology of the Nippon Veterinary and Life Science University.

2.3. Molecular analyses

Total DNA was extracted from female nematodes using theNucleoSpin Tissue Kit (TaKaRa-Bio, Japan) according to themanufacturer's protocol. Partial fragments of ITS2, 28S and COX1 wereamplified and sequenced. The ITS2 was amplified using primers LC1and HC2, which corresponded to the conserver 3′ and 5′ ends of the5.8S-ITS2-28S regions [14]. Primers 28SrDNAF and 28SrDNAR describedby Franssen [13]were used to amplify the 28S fragment. The COX1 frag-mentwas amplified using primers BP9443COIF and BP9926COIR, whichwere designed based on COX1 sequence alignments of B. procyonis(JF951366), B. transfuga (HQ671079), B. schroederi (HQ671081), andB. ailuri (HQ671080). Primer sequences are shown in Table 1.

Polymerase chain reaction (PCR) was carried out in 20 μl volumescontaining 10× PCR buffer, 25 mM MgCl2, 2.5 mM dNTPs, 50 μM eachof the primers, 5 U/μl of TaKaRa Ex Taq polymerase (TaKaRa-Bio), ster-ilized distilled water, and 1 μl of total DNA. The reaction mixture wasfirst heated at 94 °C for 2 min, followed by 30 cycles at 40 s at 94 °C,40 s at 60 °C, 1 min at 72 °C, and ending with a final extension at72 °C for 3 min. PCR products were purified using the IllustraExoProStar (GE Healthcare Life Science, USA) according to themanufacture's protocol. The purified PCR products were then se-quenced in the ABI Genetic Analyzer (Applied Biosystems, USA) usingthe Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,USA). The same primers for amplification described abovewere used forthe forward and reverse sequencing in two separate reactions. The

Table 1Primers used in this study for PCR amplification and sequencing.

Primers Amplified regions Direction

LC1 ITS2 FHC2 ITS2 RBP9443COIF COX1 FBP9926COIR COX1 R28SrDNAF 28S F28SrDNAR 28S R

F, forward primer; R, reverse primer.

reaction consisted of 25 cycles each at 96 °C for 10 s, 50 °C for 5 s, and60 °C for 4 min. Sequence data obtained in this study are available inDDBJ under the accession No. AB901104 for ITS2, No. AB893608 for28S rDNA and AB893609 for COX1.

2.4. DNA sequence analyses

Sequence similarity was determined using BLASTN from the NationalCenter for Biotechnology Informationwebsite (http://www.ncbi.nlm.nih.gov/Blast.cgi). Newly obtained sequences were used in the phylogeneticanalyses. Sequences obtained from GenBank/DDBJ/EMBL databases areas follows: B. procyonis (28S, KC543470, AY821774, U94753; COX1,JF951366), B. columnaris (28S, KC543466-9; COX1, KC543472-5),B. schroederi (28S, JN257013; COX1, HQ671081, EU628682), B. transfuga(28S, KC543471, JN257008-11; COX1, EU628683, EU628684, EU740387,HQ671079, KC543477), B. ailuri (28S, JN257012; COX1, HQ671080),Ascaris lumbricoides (28S, AY210806; COX1, HQ704900), and Toxascarisleonine (28S, JN257000; COX1, KC293927).

Sequences were aligned using a web-based version of multi-ple alignment program (MAFFT, version 7) (http://mafft.cbrc.jp/alignment/server) [15]with the Q-INS-i setting, followed bymanual ad-justment using MacClade software, version 4.08 [16]. Analyses wereperformed using MEGA version 5.2.2 [17]. All positions with gaps ormissing data were eliminated from the dataset. The amino acid se-quences were translated using invertebrate mitochondrial code(http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c#SG5). Uncorrected p-distances were calculated for sequence pairsafter removal of insertions and deletions in 2000 bootstrap replicates.Phylogenetic trees were constructed using neighbor-joining (NJ) andmaximum likelihood (ML) methods. The best-fit model was estimatedusing the Akaike Information Criterion (AIC) and the determinedKimura two-parameter plus the gamma distribution of variable sites(G = 0.05) for 28S and Hasegawa–Kishino–Yano plus the gamma dis-tribution of variable sites (G = 0.6) for COX1. Random addition of se-quences and stepwise addition of starting trees were used in MPheuristic tree search analysis. Phylogenetic trees were evaluated usingthe bootstrap methodology based on 2000 replicates for NJ and 1000replicates for ML.

3. Results

3.1. Description of the new species

3.1.1. Baylisascaris potosis n. sp. (Figs. 1, 2)Family: Ascarididae Baird, 1853Subfamily: Ascaridinae (Baird, 1853)Genus: Baylisascaris Sprent, 1968General: Body with a thick cuticle, cylindrical, slightly tapered at

both ends. Ridges of three well-defined lips detigerous (Figs. 1a, b, 2a).Denticles sharp, equilateral triangle (Figs. 1b, 2a). The dorsal lip withdouble dorso-lateral papillae (Figs. 1b, 2a, b); the subventral lips withone double ventro-lateral and one lateral papillae and one amphid(Figs. 1b, 2a, c). Cervical alae inconspicuous but cuticular bars reachingthe surface of cuticle in lateral field at base of esophago-intestinal junc-tion (Fig. 1c).

PCR primers Reference

5′-CGACTATCGATGAAGAACGCAGC-3′ [14]5′-ATATGCTTAAGTTCAGCGGG-3′ [14]5′-TTTTTCCTCATCCTGAGGTTT-3′ In this study5′-CTCCACCATAAAGTCACACCAG-3′ In this study5′-CGAGGATTCCCTTAGTAACT-3′ [9]5′-TCGGATAGGTGGTCAACG-3′ [9]

Fig. 1.Baylisascaris potosisn. sp., adultmale. a—Dorsal viewof anterior body. b—Apical viewof lips. c— Cuticle in lateralfield at base of esophageal–intestinal junction. d— Tail. e— Lateralview of spicules. f — Unembryonated egg showing thick surface (upper right). Scales apply to all figures to the right.

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Male (based on holotype and two males): Body 121 (117–127) longby 1.14 (1.10–1.21) wide at esophagus level, 1.66 (1.60–1.75) wide atmidbody. Esophagus 4.25 (4.10–4.40) long by 0.44 (0.35–0.50) wide.Spicles 0.66 (0.61–0.72) long (Fig. 1e). Pre- and post-cloacal areas ru-gose with round anterior and posterior margins, respectively (Figs. 1d,2e, f). Pre-cloacal papillae counts averaged 47.3 (44–52), situated inslightly divergent rows with somewhat irregular space (Figs. 1d, 2e).A central papilla situated on the anterior part of pre-cloacal area(Figs. 1d, 2e). Post-cloacal papillae five pairs: the first and second pairsdouble; the third and fourth pairs single; the fifth pair phasmid locatedon the sub-ventral side of the fourth pair (Figs. 1d, 2e). In a single male,the second post-cloacal papillae not fused and appeared as two singlepapillae (Fig. 2e). Tail relatively long, 0.56 (0.54–0.60)with amucronatetermination (Figs. 1d, 2e).

Female (based on allotype and a female, otherwise stated): Body 219(214–223) long by 1.65 (1.60–1.71) wide at esophagus level, 2.71(2.51–2.90) at midbody. Esophagus 4.56 long by 0.44 wide. Vulvaopened 57 (28.2% of body length) from the tail end. Tail 1.15 (1.10–1.20). Phasmids on the sub-ventral side, less than one third distance be-tween tail end and anus (Fig. 2d).

Eggs (based on 30 specimens): Unembryonated eggs, 0.087 (0.079–0.094) long by 0.071 (0.064–0.078) wide; embryonated eggs, 0.083(0.073–0.095) long by 0.073 (0.069–0.078) wide. Egg shells finelypitted (Figs. 1f, 2g).

3.1.2. Taxonomic summaryType host: Potos flavus (Schreber, 1774) (Carnivora: Procyonidae)Type locality: The Cooperative Republic of GuyanaType materials: Holotype, adult male (NSMT-As3965); allotype,

adult female (NSMT-As3966); paratype, adult male (NSMT-As3967).Etymology: The new species is named for the type host

3.1.3. RemarksPresent new species is assigned to the genus Baylisascaris based on

the presence of rough areas in the male peri-cloacal regions, both infront of and behind the cloaca.B. potosisn. sp. has inconspicuous cervicalalaewith cuticular bars that reach the cuticle surface. These characteris-tics separate B. potosis n. sp. from B. transfuga, B. melis, B. ailuri, andB. tasmaniensis having salient cervical alae [1,10,18–20]. B. potosisn. sp. differs from B. schroederi by the number of precloacal papillae

Fig. 2. Scanning electron microscopy of B. potosis n. sp. a — Apical view of male head. b— Dorsal view of dorsal lip of male. c — Dorsal view of subventral lip of male. d — Ventral view offemale posterior part. e — Ventral view of male posterior part. f — Posterior part of rugose pericloacal area. g — egg. Scale bars: a, e = 100 μm, b, c = 50 μm, d = 200 μm, f, g = 20 μm.Abbreviations: DL, dorsal lip; SVL, subventral lip; DLP, double lateral papillae; AM, amphidial pore; LP, lateral papillae; DVP, double ventral papillae; A, anus; PH, phasmidial pore;PCP, Precloacal central papillae; SP, spicule.

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(44–52 vs. 65–70) and inconspicuous denticles in B. schroederi [1,10,11].B. potosis n. sp. differs from B. devosi and B. laevis in the shape of the pos-terior margin of the rugose pericloacal area [21,22]. Male B. potosis n. sp.has phasmids at the most posterior end of the postcloacal papillae.These characteristics separate B. potosis n. sp. from B. procyonis andB. columnaris, which have phasmid located between and somewhatme-dial to the two postcloacal papillae [23–27].

Overstreet first described B. procyonis obtained from kinkajous inColombia [7]. He recognized marked differences between B. procyonisfrom kinkajou and those from raccoons, such as the morphology ofthe posterior margin of the rugose pericloacal area and phasmid loca-tion; however, he considered that these differences were not sufficient-ly quantified to differentiate species. The three B. potosis males used inour description possessed a round posterior margin in rugose post-cloacal area. On the other hand, the single degenerated male from kin-kajous showed pointed posterior margins (data not shown). Presum-ably, the morphological change observed by Overstreet and theauthors in the single male might be due to a postmortal degradation.

3.2. Phylogenetic analyses

ITS2 was obtained from a female B. potosis n. sp. The 434-bpamplicon had 100% identity with the sequence of Baylisascaris sp.(KF680774) isolated from kinkajou in our previous study [8].

28S rDNA was obtained from a female B. potosis n. sp. The 702-bpamplicon was 48.7% AT. BLAST analysis showed 99% identity withB. columnaris (KC543466-9) and B. procyonis (KC543470, U94753,AY821774). Compared to other Baylisascaris species available in theGenBank/DDBJ/EMBL databases, there were a total of 26 variable

positions in the 28S sequence (18 parsimony-informative and 8 single-ton sites) in the 702-bp pairwise alignment. Interspecific p-distancebetween B. potosis n. sp. and other species ranged from 0.012(B. columnaris) to 0.025 (B. transfuga and B. ailuri).

COX1 sequences were obtained from two B. potosis n. sp. specimens(female and male). There were no substitutions between these two se-quences. The length andAT content of COX1 sequenceswere 406bp and67.0%, respectively. BLAST analysis showed that B. potosis shared 95%identity with previously reported COX1 sequences from B. columnaris(KC543472-5) and B. procyonis (JF951366). When comparing theCOX1 sequence of B. potosis n. sp. to the other Baylisascaris species avail-able in GenBank/DDBJ/EMBL databases, a total of 50 variable positions(34 parsimony-informative and 16 singleton sites) appeared in the402-bp pairwise alignment. The amino acid sequence alignmentwas 134 aa long. B. potosis and P. procyonis differed at 17 aminoacids,while B. potosis and B. columnaris differed at 19 amino acids. Inter-specific p-distance between B. potosis n. sp. and other Baylisascarisspecies ranged from 0.058 (B. columnaris and B. procyonis) to 0.096(B. schroederi).

Phylogenetic trees based on 28S and COX1 partial sequences wereobtained by comparing sequences of B. potosis n. sp. and selectedBaylisascaris nematode sequences in the GenBank/DDBJ/EMBL data-bases. Phylogenetic analyses usingNJ andML showed similar tree topol-ogy (Fig. 3). Species of Baylisascaris assembled in a well-supportedmonophyletic clade. Phylogenetic trees constructed using 28S andCOX1 partial sequences confirmed B. potosis n. sp. to be within thewell-supported clade containing B. procyonis and B. columnaris. The re-sults obtained from phylogenetic analyses support that B. potosis n. sp.is a distinct species.

Fig. 3. Phylogenetic tree based on 28S rDNA (a) and COX1 (b) sequences of Baylisascaris spp., showing the position of B. potosis n. sp. inferred by NJ and ML. Numbers at nodes representbootstrap values (NJ/ML).

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4. Discussion

According to Sprent (1968, 1970) [1,10], Baylisascaris species aremorphologically divided into three groups. The first, group 1, containsB. transfuga, B. melis, and B. tasmaniensis; members of this group arelarger and with conspicuous cervical alae. B. ailuri, with conspicuouscervical alae, would also be included in this group. Group 2 containsB. procyonis, B. columnaris, B. devosi, and B. laevis; these members aresmaller, with inconspicuous cervical alae. Finally, Group 3 is comprisedof only B. schroederi, where the female has a longer tail and the malemore postcloacal papillae; the cervical alae are also inconspicuous.Based on these morphological characteristics B. potosis n. sp. belongsto Group 2.

Although themorphological characteristics of B. potosis n. sp. do notgenerally distinguish it from B. procyonis and B. columnaris, importantdiagnostic characteristics, such as male phasmid location, were identi-fied. In order to overcome the inadequacy of morphological analysesfor reliable differentiation of this new Baylisascaris species, a major ob-jective of this study was the development of molecular tools for reliableidentification of B. potosisn. sp. Phylogenetic trees based on different ap-proaches (NJ andML) indicated twomajor Baylisascaris cladeswith high

bootstrap support. The 28S tree showed a close relationship betweenB. schroederi and B. ailuri and than to B. transfuga. However, in theCOX1 tree, B. ailuri was more closely related to B. transfuga thanto B. schroederi. The COX1 tree was consistent with a recent study of12S rDNA [12] and amino acid sequences from 12 mitochondrialgenes [2,28]. In the present study, both trees demonstrate thatB. potosis was more closely related to B. procyonis and B. columnaristhan to other Baylisascaris species. Given that B. potosis isolated fromkinkajous is genetically and morphologically closely related toB. procyonis, there exists a potential risk for human infection withB. potosis.

Acknowledgments

The authors would like to thank the anonymous reviewers for theirvaluable comments and suggestions to improve the quality of the paper.We also thank Department of Veterinary Parasitology, Nippon Veteri-nary and Life Science University for use of SEM. This work was support-ed by a Grant-in-Aid for Research on Emerging and Re-emergingInfectious Diseases from the Ministry of Health, Labor and Welfare ofJapan (H24-Shinko-Ippan-006).

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